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The research presented in this thesis reflects advances in the adaptation and application of NMR crystallography – the synergistic combination of X-ray crystallography, NMR spectroscopy, and computational methods – to the active sites of enzymes. The goal is to construct highly detailed and chemically rich structures of the enzyme active site. The direct targets of this work are kinetically competent, quasi-stable intermediates along the reaction pathway of the pyridoxal-5’-phosphate dependent enzyme tryptophan synthase. NMR crystallography relies on the availability of both X-ray crystal structures and NMR data. This study makes use of multiple NMR restraints, including13C, 15N, and 31P NMR chemical shifts measured by collaborators in the Mueller group, and adds to these by including 17O as an active site reporter. The acid-base catalysis featured in many enzyme mechanisms relies on oxygen as a key atomic species in amphiprotic functional groups, making it a potentially important probe nucleus for NMR crystallography in enzyme active sites. Yet oxygen is not considered a standard biological NMR probe given the perceived difficulties related to its quadrupolar nuclear status. In this work we utilize solution state 17O Quadrupole Central Transition NMR to directly measure 17O chemical shifts and probe ionization states of enzyme bound intermediates.The NMR shifts are combined with X-ray crystallography to build computational models of the active site consistent with both the NMR and X-ray data. This thesis provides an exposition of the synergy of the NMR crystallographic approach applied to important intermediates of tryptophan synthase’s catalytic cycle. The results of this approach demonstrate the unprecedented level of structural detail that can be revealed, most remarkably for proton locations, within the active site. This is highlighted in particular by the characterization of proton mediated tautomerization in the “quinonoid/carbanion” intermediate, where first more...

The research presented in this thesis reflects advances in the adaptation and application of NMR crystallography – the synergistic combination of X-ray crystallography, NMR spectroscopy, and computational methods – to the active sites of enzymes. The goal is to construct highly detailed and chemically rich structures of the enzyme active site. The direct targets of this work are kinetically competent, quasi-stable intermediates along the reaction pathway of the pyridoxal-5’-phosphate dependent enzyme tryptophan synthase. NMR crystallography relies on the availability of both X-ray crystal structures and NMR data. This study makes use of multiple NMR restraints, including13C, 15N, and 31P NMR chemical shifts measured by collaborators in the Mueller group, and adds to these by including 17O as an active site reporter. The acid-base catalysis featured in many enzyme mechanisms relies on oxygen as a key atomic species in amphiprotic functional groups, making it a potentially important probe nucleus for NMR crystallography in enzyme active sites. Yet oxygen is not considered a standard biological NMR probe given the perceived difficulties related to its quadrupolar nuclear status. In this work we utilize solution state 17O Quadrupole Central Transition NMR to directly measure 17O chemical shifts and probe ionization states of enzyme bound intermediates.The NMR shifts are combined with X-ray crystallography to build computational models of the active site consistent with both the NMR and X-ray data. This thesis provides an exposition of the synergy of the NMR crystallographic approach applied to important intermediates of tryptophan synthase’s catalytic cycle. The results of this approach demonstrate the unprecedented level of structural detail that can be revealed, most remarkably for proton locations, within the active site. This is highlighted in particular by the characterization of proton mediated tautomerization in the “quinonoid/carbanion” intermediate, where first more...

The research presented in this thesis reflects advances in the adaptation and application of NMR crystallography – the synergistic combination of X-ray crystallography, NMR spectroscopy, and computational methods – to the active sites of enzymes. The goal is to construct highly detailed and chemically rich structures of the enzyme active site. The direct targets of this work are kinetically competent, quasi-stable intermediates along the reaction pathway of the pyridoxal-5’-phosphate dependent enzyme tryptophan synthase. NMR crystallography relies on the availability of both X-ray crystal structures and NMR data. This study makes use of multiple NMR restraints, including13C, 15N, and 31P NMR chemical shifts measured by collaborators in the Mueller group, and adds to these by including 17O as an active site reporter. The acid-base catalysis featured in many enzyme mechanisms relies on oxygen as a key atomic species in amphiprotic functional groups, making it a potentially important probe nucleus for NMR crystallography in enzyme active sites. Yet oxygen is not considered a standard biological NMR probe given the perceived difficulties related to its quadrupolar nuclear status. In this work we utilize solution state 17O Quadrupole Central Transition NMR to directly measure 17O chemical shifts and probe ionization states of enzyme bound intermediates.The NMR shifts are combined with X-ray crystallography to build computational models of the active site consistent with both the NMR and X-ray data. This thesis provides an exposition of the synergy of the NMR crystallographic approach applied to important intermediates of tryptophan synthase’s catalytic cycle. The results of this approach demonstrate the unprecedented level of structural detail that can be revealed, most remarkably for proton locations, within the active site. This is highlighted in particular by the characterization of proton mediated tautomerization in the “quinonoid/carbanion” intermediate, where first more...

Perchlorate in the environment is derived from both synthetic and natural sources. Synthetic perchlorate salts have been widely used as oxidants by the military and aerospace industry. A variety of commercial products also contain synthetic perchlorate. In addition to synthetic sources, natural perchlorate from both Chilean fertilizers and indigenous sources represents a potentially large source of perchlorate in groundwater and drinking water in the US. During this ESTCP project, chlorine and oxygen isotope analysis was utilized to both distinguish perchlorate sources and to document perchlorate biodegradation. Chlorine and oxygen isotopic analyses of perchlorate provide the primary direct approach whereby different sources of perchlorate can be distinguished from each other. Other isotopic, chemical, and geochemical techniques that can be applied in conjunction with isotopic analyses of perchlorate to provide supporting data in forensic studies are also described., Prepared in collaboration with the Oak Ridge National Lab, Oak Ridge, TN, the Univ of Illinois, Chicago, and Shaw Environmental, Inc. more...

Industrial nitrogen (N) emissions in the Athabasca oil sands region (AOSR), Alberta, Canada, affect nitrate (NO3) and ammonium (NH4) deposition rates in close vicinity of industrial emitters. NO3eN and NH4eN open field and throughfall deposition rates were determined at various sites between 3 km and 113 km distance to the main oil sand operations between May 2008 and May 2009. NO3 and NH4 were analyzed for d15NeNO3, d18OeNO3, D17OeNO3 and d15NeNH4. Marked differences in the d18O and D17O values between industrial emissions and background deposition allowed for the estimation of minimum industrial contributions to atmospheric NO3 deposition. d15NeNH4 values also allowed for estimates of industrial contributions to atmospheric NH4 deposition. Results revealed that particularly sites withinw30 km radius from the main oil sands developments are significantly affected by industrial contributions to atmospheric NO3 and NH4 deposition. more...

Nitrate is a common groundwater contaminant. Due to adverse health effects, waters above the Maximum Contaminant Level (MCL) of 10 mg NO3-N/L or 0.71 mmols/L, are banned from domestic consumption by the EPA. Studies have measured elevated nitrate concentrations in arid land soils and groundwater around the world. These elevated concentrations could be detrimental to the environment and to human health. Thus, it is important to consider the different sources and processes affecting nitrate concentrations Here, a novel triple isotope system approach was employed, coupling δ17O with δ18O and δ15N of nitrate to determine the sources (atmospheric, terrestrial, fertilizer, wastewater) and removal processes influencing nitrate concentrations in the Tucson basin groundwater system. Results show low groundwater nitrate concentrations (0.2 mmols/L) where wastewater was not a predominant source of water, versus high concentrations (1 mmols/L) above the MCL in groundwaters where wastewater was the dominant water source. Furthermore, groundwater up to 1.6 Km away from the wastewater stream was contaminated with effluent recharge waters. In addition, denitrification was inferred from δ18O and δ15N data with this inference reinforced by δ17O data and δ15N enrichments up to 26. Finally, low atmospheric nitrate was measured in groundwater, representing up to 6% of total nitrate. The triple isotope approach studied here is ideal for determining the proportion of atmospheric nitrate versus other terrestrial nitrate sources and the significance of nitrate removal processes. more...

Nitrate is a common groundwater contaminant. Due to adverse health effects, waters above the Maximum Contaminant Level (MCL) of 10 mg NO3-N/L or 0.71 mmols/L, are banned from domestic consumption by the EPA. Studies have measured elevated nitrate concentrations in arid land soils and groundwater around the world. These elevated concentrations could be detrimental to the environment and to human health. Thus, it is important to consider the different sources and processes affecting nitrate concentrations Here, a novel triple isotope system approach was employed, coupling δ17O with δ18O and δ15N of nitrate to determine the sources (atmospheric, terrestrial, fertilizer, wastewater) and removal processes influencing nitrate concentrations in the Tucson basin groundwater system. Results show low groundwater nitrate concentrations (0.2 mmols/L) where wastewater was not a predominant source of water, versus high concentrations (1 mmols/L) above the MCL in groundwaters where wastewater was the dominant water source. Furthermore, groundwater up to 1.6 Km away from the wastewater stream was contaminated with effluent recharge waters. In addition, denitrification was inferred from δ18O and δ15N data with this inference reinforced by δ17O data and δ15N enrichments up to 26. Finally, low atmospheric nitrate was measured in groundwater, representing up to 6% of total nitrate. The triple isotope approach studied here is ideal for determining the proportion of atmospheric nitrate versus other terrestrial nitrate sources and the significance of nitrate removal processes. more...

We have used the isotope dilution technique to study changes in the body composition of a migratory shorebird species (Red Knot, Calidris canutus) through an assessment of the amount of body water in it. Birds were quantitatively injected with a dose of water with elevated concentrations of H-2, O-17, and O-18. Thereafter, blood samples were taken and distilled. The resulting water samples were analysed using an isotope ratio mass spectrometry (for H-2 and O-18 only) and a stable isotope ratio infrared laser spectrometry (H-2, O-17, and O-18) to yield estimates of the amount of body water in the birds, which in turn could be correlated to the amount of body fat. Here, we validate laser spectrometry against mass spectrometry and show that all three isotopes may be used for body water determinations. This opens the way to the extension of the doubly labelled water method, used for the determination of energy expenditure, to a triply labelled water method, incorporating an evaporative water loss correction on a subject-by-subject basis or, alternatively, the reduction of the analytical errors by statistically combining the O-17 and O-18 measurements. more...